High density lipoprotein (HDL) and its major protein constituent, apolipoprotein (apo)A-l, play critical roles in the prevention of human cardiovascular disease, which claims nearly a million lives in the United States every year. Unfortunately, there are still many unanswered questions about the molecular basis for the cardio-protective effects of HDL. A prominent obstacle in the way of a detailed understanding of these effects is the lack of information on the structure of apoA-l in HDL particles that exist in human plasma. We propose to test the hypothesis that the structure of apoA-l in spherical plasma HDL particles is related to that found in simple HDL particles that can be created and studied in detail in vitro. In the initial funding period, we used cross-linking chemistry and high-resolution mass spectrometry to generate detailed models of apoA-l in reconstituted particles of various size and shape. In this continuation application, we propose to extend these models to real HDL particles isolated from human plasma. Using the structures we have already worked out, we will monitor the conformation of apoA-l as the complexity of the particles is systematically increased. As a first step, the effects of introducing apolipoprotein A-ll, the second most common protein constituent of HDL, on the structure of apoA-l will be evaluated. We will then monitor the structure of apoA-l and A-ll in spherical particles generated with lipid fractions isolated from plasma HDL. Finally, true human plasma particles will be used to test our models. We believe that this will be the most comprehensive study of apoA-l and apoA-ll structure in authentic human HDL particles ever attempted. We will also study the functional consequences of a shift in apoA-l molecular registry in discoidal and spherical complexes that may have exciting implications for how HDL composition modulates its metabolism. It is clear that the nature of plasma HDL precludes the use of NMR and X-ray crystallography for detailed structural studies, at least with current technology. However, our mass spectrometry approach is not limited by the requirement of a homogeneous sample and is thus the best available for addressing this important and complex problem. We believe that the information from these studies will provide the basis for future highly targeted mutagenesis strategies designed to dissect out the protective functions of apoA-l, perhaps leading to therapies for enhancing the protective effects of HDL.